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Development of a robust transient expression screening system in protoplasts of Cannabis
In Vitro Cellular & Developmental Biology - Plant ( IF 2.6 ) Pub Date : 2021-05-17 , DOI: 10.1007/s11627-021-10178-0
L. Matchett-Oates , Ehab Mohamaden , G. C. Spangenberg , N. O. I. Cogan

Transient expression systems in mesophyll protoplasts have been utilised in many plant species as an indispensable tool for gene function analysis and efficacious genome editing constructs. However, such a system has not been developed in Cannabis due to the recalcitrant nature of the plant to tissue culture as well as its illegal status for many years. In this study, young expanding leaves from aseptic in vitro Cannabis explants were used for protoplast isolation. Factorial designs were used to optimise variables in viable protoplast isolation and transient expression of GFP, with a range analyses performed to determine, and quantify, significantly impacting variables. Viable protoplast yields as high as 5.7 × 106 were achieved with 2.5% (w/v) Cellulase R-10, 0.3% (w/v) Macerozyme R-10 and 0.7 M mannitol, incubated for 16 h. As indicated by the transient expression of GFP, efficiency reached 23.2% with 30 μg plasmid, 50% PEG, 1 × 106 protoplasts and a transfection duration of 20 min. Application of the optimised protocol for protoplast isolation was successfully evaluated on three subsequent unrelated genotypes to highlight the robustness and broad applicability of the developed technique.



中文翻译:

大麻原生质体中强大的瞬时表达筛选系统的开发

叶肉原生质体中的瞬时表达系统已在许多植物物种中用作基因功能分析和有效的基因组编辑构建体的必不可少的工具。但是,由于该植物对组织培养的顽固性及其多年以来的非法地位,因此尚未在大麻中开发这种系统。在这项研究中,来自无菌体外大麻外植体的年轻膨胀叶片用于原生质体分离。析因设计用于优化可行的原生质体分离和GFP瞬时表达中的变量,并进行范围分析以确定和量化显着影响的变量。活原生质体的产率高达5.7×10 6用2.5%获得(w / v的)纤维素酶R- 10、0.3 %(w / v)Macerozyme R-10和0.7 M甘露醇,孵育16小时。如GFP的瞬时表达所示,使用30μg质粒,50%PEG,1×10 6原生质体和20分钟的转染持续时间,效率达到23.2%。优化的协议用于原生质体分离的应用已成功地评估了三个随后无关的基因型,以突出所开发技术的鲁棒性和广泛的适用性。

更新日期:2021-05-18
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